DLC-1 expression is lost in lung and other cancers and ectopic re-expression of DLC-1 impairs the transformed and tumorigenic growth of DLC-1-deficient tumor cell lines. Thus, DLC-1 exhibits properties of a tumor suppressor. DLC-1 encodes a GTPase activating protein (GAP) and negative regulator of Ras homologous (Rho) small GTPases. Constitutive activation of Rho GTPases causes growth transformation and promotes tumor cell invasion, metastasis and angiogenesis. Therefore, we hypothesize that the loss of DLC-1 function may result in persistent Rho GTPase activation and promotion of NSCLC oncogenesis. However, in addition to a RhoGAP catalytic domain, DLC-1 also contains START lipid-binding and SAM protein-protein interaction domains. Our recent results determined that DLC-1 suppresses NSCLC growth by both RhoGAP- dependent and -independent mechanisms. In addition to RhoA, we also determined that the RhoGAP domain regulates the biologically distinct RhoB and RhoC isoforms, as well as Cdc42, providing the basis for our studies to establish the full repertoire of Rho GTPases inactivated by DLC-1 (Aim 1). We also identified the SAM domain as an autoinhibitory domain that regulates DLC-1 RhoGAP activity. How this domain may regulate DLC-1 activity, and whether MEK-ERK-RSK protein kinase and PI3K-AKT lipid kinase signaling pathway-mediated phosphorylation of DLC-1 may regulate DLC-1 activity will be determined (Aim 3). DLC-1 is associated with focal adhesions (FAs) and this association is critical for DLC-1 tumor suppression but surprisingly, not for Rho GTPase inactivation in vivo. It has been determined recently that DLC-1 association with FAs is mediated by binding to tensin proteins. We also determined that the SAM and START domains may also regulate DLC-1 subcellular localization distinct from association with focal adhesions. We found that ectopic expression of DLC-1 inactivated RhoA at the leading edge of migrating cells and inhibited tumor cell invasion in vitro. These observations provide the rationale for our studies to determine the importance of spatially-restricted DLC-1-mediated Rho GTPase inactivation for inhibition of tumor growth (Aim 3). These studies will determine if SAM, START and FA-targeted DLC-1 preferentially inactivates Rho GTPases in specific subcellular compartments important for tumor suppression. Finally, to complement our ectopic re- expression studies that show DLC-1 inhibition of tumor growth, we propose interfering RNA and dominant negative DLC-1 studies to determine the biological consequences of DLC-1 loss of expression, and to evaluate a lung tumor tissue microarray to determine if loss of DLC-1 protein expression is associated with specific genetic properties and clinical outcomes of NSCLCs (Aim 4). PUBLIC HEALTH RELEVANCE: Lung cancer remains the most common fatal cancer in men (31%) and women (28%) in the US, and NSCLC accounts for 80% of all lung cancer cases and is the leading cause of cancer mortality (http://www.cancer.org/). The most recent statistics found that lung cancer death rates were increasing at a much slower rate than in the past. Unfortunately, this modest improvement is attributed primarily to decreased smoking, rather than improved therapy. Despite recent advances in molecularly targeted therapies, treatment outcomes for advanced lung cancer remain disappointing. The recent fast-track approval of EGFR inhibitors (gefitinib and erlotinib) for advanced NSCLC that have failed conventional chemotherapy have proven effective against only ~10% of NSCLCs and their impact has been modest, increasing survival by two months (erlotinib). Furthermore, the failure of gefitinib to show a survival benefit has prompted the FDA to reverse its approval for the treatment new lung cancer patients. Thus, while a subset of patients with EGFR mutations is responsive the general consensus is that EGFR inhibitors have been a disappointment for NSCLC treatment. Therefore, new target-based treatment strategies are clearly needed in NSCLC therapy. One possible class of targets is the Ras homologous Rho small GTPases. There is considerable and growing evidence for aberrant Rho GTPase function in oncogenesis, in particular in breast, pancreatic, and head and neck carcinomas, and melanomas. However, to date, there has been surprisingly limited study of the role of aberrant Rho GTPases in NSCLC growth. We propose studies to address one key mechanism by which Rho GTPase function may be deregulated in a majority of NSCLCs, the loss of the DLC-1 tumor suppressor. Our recent evidence supports our hypothesis that loss of DLC-1 causes hyperactivation of Rho GTPases in NSCLC. Hence, we believe that our elucidation of the mechanism by which DLC-1 loss may deregulate Rho GTPases and promote NSCLC growth may define novel directions for targeted therapies for lung cancer treatment.